Research Laboratory 1: High Resolution Imaging

ADVANCED MICROSCOPY SYSTEMS AT CBST

RESEARCH LABORATORY 1 – HIGH RESOLUTION IMAGING

Atomic force microscopy:

A Veeco/Digital Instruments Bioscope II atomic force microscopy system that can be combined with all of the optical microscopesat CBST is available. This microscope represents the latest incarnation of a combined optical microscope and atomic force microscope. It is supported by an Olympus IX71 microscope frame with differential interference contrast for live cell viewing of unstained samples. The microscope stage supports glass-bottom culture dishes with up to 60 mm diameter, and incorporates a sample heating, perfusion, and gas maintenance chamber for prolonged live cell experiments. Samples can be positioned within the 100µm x 100 µm x 20 µm scan range of the piezo scanner by joystick control of stepper motors. The AFM tip can be positioned in the center of the optical microscope through manual micro-actuators. The digital AFM controller supports up to 8 arbitrarily assignable data acquisition channels. The software provides full microscope control, data acquisition, and image processing capabilities. It supports 1D, 2D, and 3D topographical imaging, point force spectroscopy, elastic modulus imaging, contact-mode, as well as tapping-mode scanning probemicroscopy.

Deconvolution fluorescence microscope:

A personalDV deconvolution fluorescence microscope (Applied Precision, LLC, Issaquah, WA) is available for rapid live and fixed cell fluorescence microscopy. Complex custom acquisition protocols can be programmed to revisit samples over a 5cm x 5cm area. The system provides image-based auto-focusing and can image samples repeatedly over several days. Excitation/emission wavelengths are freely adjustable by swapping bandpass filters in a computer-controlled motorized filter wheel. Up to six filter combinations can be used. Currently the microscope is equipped for DAPI/FITC/Rhodamine/Alexa647 fluorescence imaging. This microscope is electronically registered with the structured illumination microscope described below and can be used to survey large samples that can then be imaged at high resolution with structured illumination.

Structured illumination fluorescence microscope:

CBST is proud to own the first commercial prototype of a structured illumination microscope (OMX v2.0, Applied Precision, LLC, Issaquah, WA) is available for super-resolution wide-field fluorescence microscopy. This microscope is equipped with 3 solid state lasers operating at 405 nm, 488 nm, and 532 nm for fluorescence excitation. The system uses custom-made filters for DAPI/FITC/Rhodamine excitation, but can be extended to other filter combinations. Two electron-multiplying CCD cameras with 512x512 pixels (Photometrics Cascade II) cooled to -90 deg. C can be used for fast live cell deconvolution microscope or structured illumination imaging. A dedicated digital signal processing system controls the data acquisition and timing. Samples can be translated in the transverse and lateral dimensions by a combination of micro-positioners and piezo-electric positioners. For structured illumination microscopy the laser beams are routed through a phase grating and an interference pattern is generated at the sample position. 5 images at different grating positions are taken with the grating pattern being translated by piezoelectrics. The grating is then rotated by +/- 60 deg. and 5 more images are taken for each grating position. The image sequence is then used to reconstruct a sample’s image by utilization of software with 100 nm lateral spatial resolution and 200 nm vertical spatial resolutions.